Salts of isophosphoramide mustard and analogs thereof as anti-tumor agents

ABSTRACT

The present disclosure relates to salts and compositions of isophosphoramide mustard and isophosphoramide mustard analogs. In one embodiment the salts can be represented by the formula I: (I) wherein A +  represents an ammonium species selected from the protonated (conjugate acid) or quaternary forms of aliphatic amines and aromatic amines, including basic amino acids, heterocyclic amines, substituted and unsubstituted pyridines, guanidines and amidines; and X and Y independently represent leaving groups. Also disclosed herein are methods for making such compounds and formulating pharmaceutical compositions thereof. Methods for administering the disclosed compounds to subjects, particularly to treat hyper-proliferative disorders, also are disclosed.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant No.5R44CA083552-03 awarded by the National Cancer Institute. The governmenthas certain rights in the invention.

FIELD

This disclosure concerns salts of isophosphoramide mustard and analogsthereof. Also disclosed are pharmaceutical compositions and methods forusing such compositions to treat hyper-proliferative disorders.

BACKGROUND

Autopsies of soldiers killed by mustard gas in World War I indicatedthat sulfur mustard has a disproportionate effect on rapidly dividingcells and suggested that sulfur mustard compounds might have antitumoreffects. Indeed, early researchers attempted to treat cancer by directinjection of sulfur mustard into tumors. This research was limited bythe extreme toxicity of sulfur mustard compounds and nitrogen mustardanalogs, such as mechlorethamine, were investigated as less toxicalternatives.

In general mustard compounds exert their cytotoxic effects by alkylatingDNA, such as at the N-7 position of a guanine residue. The mechanism ofalkylation by mustard compounds is illustrated in Scheme 1. Withreference to Scheme 1, mustard compounds have an internal nucleophilethat assists in chloride displacement, by as shown for the case ofmechlorethamine, forming an aziridinium intermediate. Becausemechlorethamine has two leaving groups, the nucleophilic substitutionmechanism depicted in Scheme 1 can be repeated resulting in a DNA orprotein-DNA crosslink.

Meclilorethamine is extremely reactive and as a result is non-selective.Thousands of alkylating agents have been designed and prepared usingmechlorethamine as a model. However, few of these compounds havedemonstrated sufficient therapeutic superiority to mechlorethamine towarrant clinical trials.

Because of the lack of selectivity of most methchlorethamine analogs,prodrugs, such as phosphoramide compounds, which can be activated by thehigh concentration of phosphoramidases present in neoplastic cells, havebeen investigated. Two phophoramide alkylating agents, cyclophosphamide(CPA) and the isomeric compound Ifosfamide (Ifos) have proved to beparticularly effective.

The metabolic pathway of CPA is similar to that of Ifos (the metabolismof Ifos is illustrated in FIG. 1) and thus the two compounds sharecommon drawbacks. Perhaps most important is their dose limiting toxicitydue to hemorrhagic cystitis. The hemorrhagic cystitis is believed to beinduced by the production of acrolein during the activation of both CPAand Ifos. Acrolein is an active electrophile that reacts with thiolsunder physiological conditions, which may be responsible for its livertoxicity in the form of glutathione depletion. Finally, acrolein hasbeen demonstrated to be a teratogen and a potent mutagen, and this maybe responsible for the link between CPA treatment and serious sideeffects, such as bladder carcinoma and other malignancies.

With reference to FIG. 1, isophosphoramide mustard (LPM) is a commonmetabolite of CPA and Ifos. IPM is thought to be responsible for atleast a portion of the anti-tumor activity exhibited by CPA and Ifos.Efforts to use IPM as an anticancer agent directly have beenunsuccessful due in part to the compound's instability. IPM has beensynthesized and preliminary biological evaluations of the compound havebeen conducted, but unfortunately IPM is rod unstable to be isolated andused for human treatment.

SUMMARY OF THE DISCLOSURE

Disclosed herein are compounds of the formula

wherein A⁺ represents an ammonium species selected from the protonated(conjugate acid) or quaternary forms of aliphatic amines and aromaticamines, including basic amino acids, heterocyclic amines, substitutedand unsubstituted pyridines, guanidines and amidines; and X and Yindependently represent leaving groups.

In one embodiment, pharmaceutical compositions are disclosed thatinclude one or more of the compounds described above. In one aspect ofthis embodiment, the compositions can include one or more therapeuticagents other than those described by the formula above for use incombination therapy.

In another embodiment, methods for treating mammalian subjects, such ashuman subjects, having hyperproliferative disorders are disclosed. Suchmethods can employ one or more of the compounds and compositionsdescribed above.

In another aspect, disclosed herein are sterile pharmaceuticalcompositions of compounds of the formula

wherein X and Y independently represent leaving groups, or apharmaceutically acceptable salt thereof. Methods of preparing suchcompositions, including rendering the composition sterile by using asterile antimicrobial filter, are also described. In certainembodiments, such filtration may be performed with less than 10%decomposition of the active ingredient, preferably less than 5%, 2%, oreven less than 1% decomposition.

Also disclosed herein is a method for producing a lyophilisatecomprising a compound of the formula above. In one embodiment the methodcomprises contacting isophosphoramide mustard or an analog thereof withan amine base in the presence of water and lyophilizing the resultingmixture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme illustrating the metabolism of ifosfamide includingthe production of acrolein and isophosphoramide mustard.

FIG. 2 is a ¹H NMR spectrum of IPM.(LYS)₂ in D₂O recorded at 500 MHz.

FIG. 3 is an expanded section of the spectrum in FIG. 2.

FIG. 4 is ¹³C NMR spectrum of IPM-(LYS)₂.

DETAILED DESCRIPTION

The following explanations of terms and examples are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be understood to have thefollowing meanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance can but need not occur, and that the descriptionincludes instances where said event or circumstance occurs and instanceswhere it does not.

The term “amino acid” refers to both natural and unnatural amino acids,including α-amino acids, in their D and L stereoisomers for chiral aminoacids. Examples of basic amino acid residues include those having abasic side chain, such as an amino or guanidino group. Basic amino acidresidues include, without limitation, arginine, histidine, homoarginine,lysine, homolysine and ornithine.

The term “antibody” means an immunoglobulin, whether natural or whollyor partially synthetically produced. All derivatives thereof whichmaintain specific binding ability are also included in the term. Theterm also covers any protein having a binding domain which is homologousor largely homologous to an immunoglobulin binding domain. Theseproteins may be derived from natural sources, or partly or whollysynthetically produced. Antibodies used herein may be monoclonal orpolyclonal.

As used herein, “aliphatic amine” refers to a compound of the formulaNR¹R²R³, wherein at least one of R¹⁻³ is an aliphatic group.

The term “acyclic aliphatic amine” refers to an aliphatic amine asabove, wherein at least one of the aliphatic groups is acyclic.

The term “heterocyclic amine” refers to a compound of the formulaNR¹R²R³, wherein at least one of R¹⁻³ is a heterocyclic group or R¹, R²and/or R³ taken together with their common nitrogen atom form a ring.

I. Salts of IPM and IPM Analogs

The compounds and compositions disclosed herein include IPM and IPManalogs that are formulated with one or more equivalents of a base.Because IPM and its analogs are acid labile and are acidic, thepresently disclosed compounds offer greater stability as well as otheradvantages. The advantages of the disclosed formulations in terms ofsynthesis, stability and bioavailability will be apparent to those ofordinary skill in the art upon consideration of the present disclosure.

In one embodiment, the disclosed compounds are salts of isophosphoramidemustard or isophosphoramide mustard analogs including one or morecations. In one embodiment, the cations can be a conjugate acid of anamine base or can be a quaternary ammonium cation. Suitable counterionsfor isophosphoramide mustard and its analogs include the conjugate acids(as used herein terms that refer to amines should be understood toinclude their conjugate acids unless the context clearly indicates thatthe free amine is intended) of bases including basic amino acids,aliphatic amines, heterocyclic amines, aromatic amines, pyridines,guanidines and amidines. Of the aliphatic amines, the acyclic aliphaticamines, and cyclic and acyclic di- and tri-alkyl amines are particularlysuitable for use in the disclosed compounds. In addition, quaternaryammonium counterions are examples of suitable counterions that can beused.

Particular examples of suitable amine bases (and their correspondingammonium ions) for use in the present compounds include, withoutlimitation, pyridine, N,N-dimethylaminopyridine, diazabicyclononane,diazabicycloundecene, N-methyl-N-ethylamine, diethylamine,triethylamine, diisopropylethylamine, mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amino,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine.

In a further embodiment, the salts described above can include a secondamine or ammonium group. In one embodiment the compounds disclosedherein include more than one equivalent of an amino for each equivalentof isophosphoramide mustard or isophosphoramide mustard analog. Suchembodiments include those having non-integer ratios of amine toisophosphoramide mustard or isophosphoramide mustard analogs. In certainembodiments, the compounds have a two to one or three to one ratio ofamine to isophosphoramide mustard or an isophosphoramide mustard analog.In working embodiments salts were produced containing two equivalents ofamine base per equivalent of isophosphoramide mustard. In oneembodiment, an amine base used to form isophosphoramide mustard andisophosphoramide mustard analog salts includes more than one aminogroup; such bases can be termed “multibasic.” More specifically, certainexamples of multibasic bases that can be used have two amino groups;such compounds can be referred to as “dibasic.” For example, onesuitable dibasic molecule is N,N-dimethylaminopyridine, which includestwo basic amino groups. In a particular embodiment of a compounddisclosed herein a compound includes isophosphoramide mustard or anisophosphoramide mustard analog and one equivalent of a dibasic amine.

In one embodiment, the disclosed compounds include one or morezwitterionic bases. Examples of such bases include basic amino acids,which are zwitterionic at physiological pH.

In one embodiment the presently disclosed salts are more stable thanisophosphoramide mustard and isophosphoramide mustard analogs. Forexample, isophosphoramide mustard, following lyophilization of the purecompound, decomposes by nearly 40% during storage at −20° C. for threemonths. In contrast, the lysine salt of IPM does not exhibit anymeasurable decomposition, even after ten months under similar storageconditions.

In certain embodiments, the disclosed compounds are stabilizedisophosphoramide mustard salts or stabilized isophosphoramide saltanalogs, wherein the salt has a half-life at room temperature (e.g.,about 23° C.) in the presence of water that is greater than a half-lifeof isophosphoramide mustard in the presence of water under the sameconditions. In certain preferred such embodiments, an isophosphoramidemustard salt has a half-life that is equal to or greater than twice aslong as isophosphoramide mustard in the presence of water, morepreferably, equal to or greater than five times.

In certain embodiments, lyophilisates of disclosed compounds are morestable than a lyophilized preparation of isophosphoramide mustard. Incertain preferred such embodiments, the lyophilisate of the disclosedcompounds have a longer shelf life than a lyophilized preparation ofisophosphoramide mustard itself, preferably at least twice as long, morepreferably at least five times as long.

In certain embodiments, pharmaceutical compositions of pharmaceuticallyacceptable salts of IPM or its analogs (such as the compounds of theabove formula) are more stable than an otherwise identical compositionof isophosphoramide mustard itself (i.e., not in a salt form) underidentical conditions. In certain preferred such embodiments, thedisclosed compositions have a longer shelf life than the lyophilizedpreparation of isophosphoramide mustard, preferably at least twice aslong, more preferably at least five times as long.

Certain isophosphoramide mustard and isophosphoramide mustard analogcompounds disclosed herein include two leaving groups. Withoutlimitation to theory, it is believed that the two leaving groups aredisplaced in vivo by biomolecular nucleophiles, such as nucleic acidsand proteins, thereby cross-linking the biomolecules. The term “leavinggroup” refers to a group that can be displaced by a nucleophile. Withreference to the presently disclosed compounds, leaving group refers toa group that can be displaced to form an aziridinium intermediate, orcan be directly displaced by a biomolecular nucleophile, such as anucleic acid nucleophile, to form, for example, a 7-alkylatedguanidinium species. Examples of suitable leaving groups include thehalogens and the sulfonates (—SO₂R). In one embodiment of the disclosedisophosphoramide analog salts, the compound is a “mixed” leaving groupcompound, including two different types of leaving groups, for example ahalogen and a sulfonate or two different halogens, such as a bromide anda chloride. U.S. Pat. No. 6,197,760 to Struck teaches methods for makingsuch mixed leaving group compounds.

One embodiment of the present disclosure concernsanti-hyperproliferative agents of the formula

With reference to the formula, B can be, for each n, an independentlyselected basic molecule. In one embodiment of the formula, B can beselected from the basic amino acids, acyclic aliphatic amines, di- andtri alkyl amines, heterocyclic aliphatic amines, aromatic amines,substituted and unsubstituted pyridines, cyclic and acyclic guanidines,and cyclic and acyclic amidines. Typically, n is from 1 to about 3 suchthat the formula can include different basic molecules. With continuedreference to the formula, X and Y are leaving groups. A person ofordinary skill in the art will understand that the illustratedisophosphoramide mustard structure includes an acidic proton, and assuch exists predominantly as its conjugate base at physiological pH andin the presence of a base such as B. Likewise, B, being a basic groupexists predominantly as its conjugate acid at physiological pH and inthe presence of isophosphoramide mustard and isophosphoramide mustardanalogs. Exemplary embodiments of the disclosed compounds are depictedin Table 1.

TABLE 1

B n X Y lysine 2 Cl Cl NH₃ 2 Cl Cl cyclohexylamine 2 Cl ClN-methyl-D-glucamine 2 Cl Cl N,-N-dimethylaminopyridine 1 Cl Cl arginine2 Cl Cl lysine 2 Cl —SO₂CH₃ lysine 2 Br —SO₂CH₃

In a further embodiment, the disclosed compounds include salts ofisophosphoramide mustard. Certain examples of such isophosphoramidemustard salts can be represented by the formula

With reference to the formula above, B can be any basic group,particularly an amine. It should be recognized that the formula abovewill exist predominantly as the corresponding salt and thus can includecompounds that are represented by the formula

With reference to the formula above, such compounds also can include oneor more additional equivalents of an amine or ammonium species. Suchcompounds can be represented by the formula

wherein G represents a second ammonium or amine species. In particularexamples, G is a basic amino acid and BH⁺ represents the conjugate acidof the same or a different basic amino acid.

In one embodiment BH⁺ is the conjugate acid of G. In this embodiment,the disclosed isophosphoramide mustard salts can be represented by theformula

wherein B is an amine and BH⁺ is its conjugate acid.

In one embodiment, the compounds disclosed herein include a metalcation, such as an alkali metal cation. Examples of such cations includeLi⁺, Na⁺, K⁺ and Rb⁺ and Cs⁺. In one aspect, such examples can berepresented by the formula

wherein M⁺ represents an alkali metal cation and B is as defined above.

II. Compositions and Methods

Another aspect of the disclosure includes pharmaceutical compositions,preferably sterile pharmaceutical compositions, prepared foradministration to a subject and which include a therapeuticallyeffective amount of one or more of the currently disclosed compounds.Such sterile compositions may be prepared by passing a solution of thesalt of IPM or an analog thereof through a sterile antimicrobial filter.Such sterile compositions preferably comprise the active ingredient ofthe invention with less than 10% degradation, preferably less than 5%,2%, or even less than 1% decomposition as measured by assaying for thepresence of decomposition by-products such as phosphoric acid and itssalts and substituted ethylamines.

The compounds disclosed herein may be administered orally, topically,transdermally, parenterally, via inhalation or spray and may beadministered in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

Typically, parenteral administration of the disclosed isophosphoramidemustard salts and analogs thereof via injection is preferred. Theinhibitors may be provided in a single dosage or chronically, dependentupon the particular disease, condition of patient, toxicity of compoundand other factors as will be recognized by a person of ordinary skill inthe art.

The therapeutically effective amount of the compound or compoundsadministered can vary depending upon the desired effects and the factorsnoted above.

Pharmaceutical compositions for administration to a subject can includecarriers, thickeners, diluents, buffers, preservatives, surface activeagents and the like in addition to the molecule of choice.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. Pharmaceutical formulations caninclude additional components, such as carriers. The pharmaceuticallyacceptable carriers useful for these formulations are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of the compoundsherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

In one embodiment, a disclosed compound is formulated for administrationto a human subject. In aspect of this embodiment the pharmaceuticalcomposition includes from about 0.1 mg/mL to about 250 mg/mL, such asfrom about 20 to about 100 mg/mL of the compound of an isophosphoramidemustard salt or analog thereof.

In one aspect, certain embodiments of pharmaceutical compositions areformulated into unit dosage forms. For example such unit dosage formscan contain from about 100 mg to about 1500 mg, such as from about 200mg to about 1500 mg of a disclosed isophosphoramide mustard salt oranalog thereof per dosage unit.

It is specifically contemplated in some embodiments that the presentcompounds are delivered via an injected and/or implanted drug depot, forinstance comprising multi-vesicular liposomes such as in DepoFoam(SkyePharma, Inc. San Diego, Calif.) (see, for instance, Chamberlain etal. Arch. Neuro. 1993, 50, 261-264; Katri et al. J. Pharm. Sci. 1998,87, 1341-1346; Ye et al., J. Control Release 2000, 64, 155-166; andHowell, Cancer J. 2001, 7, 219-227).

Methods are disclosed herein for treating conditions characterized byabnormal or pathological proliferative activity or neoplasia byadministering one or more of the disclosed compounds and compositions toa subject “Neoplasia” refers to the process of abnormal and uncontrolledcell growth. Neoplasia is one example of a proliferative disorder. Theproduct of neoplasia is a neoplasm (a tumor), which is an abnormalgrowth of tissue that results from excessive cell division. A tumor thatdoes not metastasize is referred to as “benign.” A tumor that invadesthe surrounding tissue and/or can metastasize is referred to as“malignant.”

Conditions that can be treated according to the disclosed method includethose characterized by abnormal cell growth and/or differentiation, suchas cancers and other neoplastic conditions. Typical examples ofproliferative disorders that can be treated using the disclosedcompounds and compositions are listed below.

Examples of hematological tumors that can be treated using the compoundsand compositions disclosed herein include leukemias, including acuteleukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Additional examples of conditions that can be treated using thedisclosed compounds and compositions include solid tumors, such assarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma,mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, coloncarcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lungcancers, ovarian cancer, prostate cancer, hepatocellular carcinoma,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma,renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, andCNS tumors (such as a glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

In one embodiment the compounds disclosed herein superior to CPA or Ifosalone against CPA resistant tumor growth. Therefore one aspect of amethod disclosed herein includes treating a subject having a CPAresistant neoplastic condition with an isophosphoramide mustard salt oranalog thereof disclosed herein.

In one embodiment of the method a subject is administered from about 0.2mg/kg/day to about 20 mg/kg/day of a disclosed isophosphoramide mustardsalt or analog thereof. For example, from about 0.5 to about 10mg/kg/day, such as from about 1 to about 7.5 mg/kg/day of a disclosedcompound can be administered to a subject.

In another embodiment of the method a subject is administered from about10 to about 700 mg/m²/day, such as from about 20 to about 400 mg/m²/dayor from about 100 to about 500 mg/m²/day. For example, from about 30 toabout 100 mg/m²/day, such as from about 40 to about 90 mg/m²/day of acompound disclosed herein.

In one embodiment of the method for treating hyper-proliferativedisorders disclosed herein, a disclosed compound is administered to asubject on a multiple daily dosing schedule. In such embodiments thecompound is administered on at least two days and on as many as fivedifferent days. In one aspect of multiple daily dosing schedules, thecompound is administered to the subject on consecutive days, such asfrom two to five consecutive days.

In one embodiment of the method one or more additional therapeuticagents is administered to a subject in addition to the presentlydisclosed compounds and compositions. For example, additionaltherapeutic agents can that can be used include microtubule bindingagents, DNA intercalators or cross-linkers, DNA synthesis inhibitors,DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzymeinhibitors, gene regulators, and/or angiogenesis inhibitors.

“Microtubule binding agent” refers to an agent that interacts withtubulin to stabilize or destabilize microtubule formation therebyinhibiting cell division. Examples of microtubule binding agents thatcan be used in conjunction with the presently disclosed isophosphoramidemustard salts and analogs thereof include, without limitation,paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine),the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxinand rhizoxin. Analogs and derivatives of such compounds also can be usedand will be known to those of ordinary skill in the art. For example,suitable epothilones and epothilone analogs for incorporation into thepresent compounds are described in International Publication No. WO2004/018478, which is incorporated herein by reference. Taxoids, such aspaclitaxel and docetaxel are currently believed to be particularlyuseful as therapeutic agents in the presently disclosed compounds.Examples of additional useful taxoids, including analogs of paclitaxelare taught by U.S. Pat. No. 6,610,860 to Holton, U.S. Pat. No. 5,530,020to Gurram et al. and U.S. Pat. No. 5,912,264 to Wittman et al. Each ofthese patents is incorporated herein by reference.

Suitable DNA and/or RNA transcription regulators, including, withoutlimitation, actinomycin D, daunorubicin, doxorubicin and derivatives andanalogs thereof also are suitable for use in combination with thepresently disclosed compounds. DNA intercalators and cross-linkingagents that can be incorporated into the disclosed compounds include,without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins,such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide andderivatives and analogs thereof.

DNA synthesis inhibitors suitable for use as therapeutic agents include,without limitation, methotrexate, 5-fluoro-5′-deoxyuridine,5-fluorouracil and analogs thereof.

Examples of suitable enzyme inhibitors for use in combination with thepresently disclosed compounds include, without limitation, camptothecin,etoposide, formestane, trichostatin and derivatives and analogs thereof.

Suitable therapeutics for use with the presently disclosed compoundsthat affect gene regulation include agents that result in increased ordecreased expression of one or more genes, such as, without limitation,raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen,4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.

The term “angiogenesis inhibitor” is used herein, to mean a moleculeincluding, but not limited to, biomolecules, such as peptides, proteins,enzymes, polysaccharides, oligonucleotides, DNA, RNA, recombinantvectors, and small molecules that function to inhibit blood vesselgrowth. Angiogenesis is implicated in certain pathological processes,such as those involved in disorders such as diabetic retinopathy,chronic inflammatory diseases, rheumatoid arthritis, dermatitis,psoriasis, stomach ulcers, and most types of human solid tumors.

Angiogenesis inhibitors are known in the art and examples of suitableangiogenesis inhibitors include, without limitation, angiostatin K1-3,staurosporine, genistein, fumagillin, medroxyprogesterone, suramin,interferon-alpha, metalloproteinase inhibitors, platelet factor 4,somatostatin, thromobospondin, endostatin, thalidomide, and derivativesand analogs thereof.

Other therapeutic agents, particularly anti-tumor agents, that may ormay not fall under one or more of the classifications above, also aresuitable for administration in combination with the presently disclosedcompounds. By way of example, such agents include adriamycin, apigenin,rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.

III. Examples

The foregoing disclosure is further explained by the followingnon-limiting examples.

Example 1

This example describes the synthesis of the phenyl ester of IPMaccording to the scheme.

To a 5 L 3 neck round bottom flask equipped with a mechanical stirrer, a500 mL drip funnel and a calcium chloride drying tube 2-chloroethylaminehydrochloride (116 g; 1.0 mol) is suspended in 1200 mL methylenechloride and stirred in an ice water bath. When the temperature fell to5° C., phenyldichlorophosphonate (105.5 g; 0.5 mol) (commerciallyavailable from Aldrich, Milwaukee, Wis.) was added. Triethylamine (202g, 2 mol) was dripped in slowly at 1 drop per second; the temperaturedid not exceed 5° C.: The mixture is allowed to stir overnight. Thefollowing day, 200 mL concentrated hydrochloric acid (12 M) was mixedwith 1800 mL water. To the reaction mixture was slowly added 200 mL ofthe acid solution. The mixture became clear and was transferred to a 2 Lseparatory funnel and the organic and aqueous layers separated. Theorganic layer was extracted with the acid solution 9×200 mL followed bywater 1×200 mL. The organic layer was then separated and dried oversodium sulfate and filtered. The methylene chloride was then evaporatedunder reduced pressure and the oil residue was dissolved in 40 mL ethylacetate and 60 mL hexane was slowly added with stirring; it was thencovered with Parafilm and kept at 5° C. in a freezer overnight. Thefollowing day the white crystals were suction filtered and washed with100 mL cold hexane and then allowed to air dry. The mother liquid waskept in the freezer for 9 hours and a second crop of crystals formed andthese were allowed to air dry. A third crop of crystals formed onfreezing the mother liquor from the second crop overnight and these wereair-dried. The combined crops had a yield of 117.3 grams; 0.39 mol. Theyield was 82%; m.p. 53-55; Anal. Calcd for C₁₀H₁₅Cl₂N₂O₂P (F.W. 297.13)C, 40.44%; H, 5.09%; N, 9.43%. Found C, 39.7%; H, 4.97%; N, 9.00%.

Example 2

This example describes the synthesis of IPM(N,N′-di(2-chloroethyl)phosphorodiamidic acid) from the IPM phenyl esterdescribed in example 1.

The white solid ester from example 1 (0.39 mol) was dissolved in 100 mLof 95% ethanol and added to a Parr flask and 2.5 g PtO₂ was added. Thesuspension was hydrogenated at 50 PSI; two hours later the hydrogenationwas stopped and 2.5 g PtO₂ was cautiously added with stirring.Hydrogenation was resumed at 50 PSI for two hours. It was then stopped,brought to ambient pressure and heated on a hot plate with magneticstirring. When the suspension was boiling it was suction filteredimmediately through a 5.5 cm suction funnel with two filter papers andthe supernatant stored at 5° C. for two hours; the catalyst is saved andadded to the Parr flask and stored in a freezer overnight. The whitesolid that formed was suction filtered through a 9 cm suction funnel andsaved in a pesticide free jar, the mother liquor was added to the Parrflask and 1.25 grams PtO₂ was added it was hydrogenated at 50 PSI fortwo hours. It was stopped, heated and filtered as before and the motherliquor kept in the freezer overnight. The white crystals that formedwere suctioned filtered and combined with the first crop. The motherliquor is collected in the Parr flask with the used catalyst and anadditional 1.25 grams of PtO₂ is added and hydrogenation is resumed at50 PSI for 2 hours. It was then stopped, heated and filtered to producea third crop, which was combined with crops 1 and 2. The combined cropswere stirred in 150 mL of acetone for 30 minutes then stored at 5° C.for two hours and then filtered and stored in a vacuum desiccator fortwo hours. The yield was 38 g; 0.17 mol; 44% yield; mp(corr) 112-114.Anal. Calcd for C₄H₁₁N₂O₂PCl₂ (F.W. 221.11) C, 21.73%; H, 5.01%; N,12.67%. Found C, 22.12%; H, 5.02%; N, 12.23%.

Example 3

This example describes the preparation of IPM lysine salt from IPMproduced according to example 2. The L-lysine was weighed (26.4 g) andthe water measured (6 L). The L-lysine is added to the water withstirring @2-8° C. The bulk drug substance, IPM, is weighed (20 g) andadded slowly with stirring @2-8° C. to the lysine solution.

Once dissolved at 2-8° C., the solution is passed through a sterileantimicrobial filter (0.22 microns). The solution is maintained at 2-8°C. and dispersed into vials under sterile conditions.

The dissolved product is then lyophilized under the followingconditions.

Time Temperature (Hours) Action (° C.) 0-1 Loading 0 −45 1-7 Hold −45 7-34 Primary Dry −25 34-55 Secondary Dry −10 55-76 Secondary Dry 0 76+Nitrogen purge 0Alternatively, the dissolved product may be lyophilized under thefollowing conditions.

Time Temperature (Hours) Action (° C.)  0-0.5 Loading 0 −45 0.5-6.5 Hold−45 6.5-33  Primary Dry −25 33-54 Primary Dry −10 54-95 Secondary Dry 095+ Nitrogen purge 0

The vials are capped under sterile conditions according to standardoperating procedure. The lyophilized IPM lysine salt is packaged inunprinted glass vials with crimped rubber sealed caps. Thiscontainer/closure system doer not include a liner. Negative ionelectrospray mass spectrometry revealed characteristic peaks forIPM.(LYS)₂ at M=219.0, 441.0 (dimer) and 662.7 mass units (trimer). The1H NMR and ¹³C NMR spectra of IPM.(LYS)₂ in D₂O are provided as FIGS.2-4.

The cyclohexyl amine and ammonium salts of IPM were prepared asdescribed above for the lysine salt. Each of these salts was isolatedhaving a 2:1 stoichiometry of amine:IPM.

Example 4

This example describes the evaluation of IPM against several differentcancer cell lines implanted in mice. The mice tolerated intraperitoneal(IP) and intravenous (IV) treatment with IPM well in each study; theonly toxicities are organ pathologies observed at autopsy that wereassociated with the induced tumors.

First, IPM was evaluated against two L1210 variants, L1210/0 andL1210/CPA cell lines implanted in mice, as compared with Ifos. Thedosages for IPM were 50% of the dose for Ifos. ILS was observed for allthree agents in the L1210/0 treated groups. However, for the L1210/CPAmodel, the IPM treatment demonstrated superiority over the other twoarms (Ifos vs CPA). In the CPA resistant tumor line, the IPM treatedanimals had a two-fold increase in survival with a tumor burdenreduction of 7. For the L1210/0 tumor model, IPM was equally active toCPA and Ifos, but at a lower dose. This demonstrates that CPA resistantcells are not cross-resistant to IPM. The results of this study arerecorded below in Table 2.

TABLE 2 Activity of isophosphoramide mustard against L1210/0 andL1210/CPA leukaemias (Optimal response at #LD₁₀ dose, from dose-responsestudy) L1210/CPA† L1210/0† Tumor burden at start of Tumor burden atstart of R_(x) = 6.0 × 10⁷ cells R_(x) = 8.5 × 10⁷ cells Net log₁₀ Netlog₁₀ Reduction Day 60 Reduction in % ILS in tumor Sur- % ILS tumorburden Day 60 (dying burden Dosage* vivors/ (dying after survivors/ miceafter Agent (mg kg⁻¹) total mice only) therapy‡ total only) therapy‡Cyclophosphamide 200 0/10 +107 7 0/10 +57 4 Ifosfamide 431 0/10 +185 80/10 +85 5 289 0/9  +114 8 0/10 +57 4 Isophosphoramide 100 0/10 +128 81/10 +114 7 mustard *Treatment: IP; day 2 only; highest non-toxic dose(LD₁₀ or less) in a range of doses. †IP; 10⁶ cells, in male CDF₁ mice.

A second study demonstrates the inhibition of Lewis lung carcinoma byIPM in mice implanted with Lewis lung carcinoma tumors. Single secondday IP dosings with CPA, Ifos, PM and IPM to mice bearing Lewis Lungcarcinoma revealed that IPM yielded 6/10 tumor free survivors, ascompared to 7/10 for Ifos and 5/10 for CPA at equitoxic, equal doses.The single dose schedule was used for each agent and the activitiesnoted (T-C) were the same between all four agents.

The results of this study, recorded in Table 3, demonstrate that IPM iseffective against Lewis lung carcinoma.

TABLE 3 Response of Lewis Lung Carcinoma to Isophosphoramide MustardImplant Size: 20-30 mg; Implant Site: s.c.; Drug Treatment: IP Highestnon- Log toxic dosage Tumor-free kill Agent Schedule (mg kg⁻¹/dose)survivors T-C*† % ILS‡ totalδ Cylophosphamide Day 2 200 5/10 27 68 >6.8Single dose Ifosfamide Day 2 300 7/10 18 55 >4.5 Single dosePhosphoramide Day 2 200 0/10 4.9 15 1.2 mustard Single dose Day 2 300/10 6.1 17 1.5 Q5 min × 7 Isophosphoramide Day 2 100 6/10 8.4 34 >2.1mustard Single dose *Tumor growth delay (T-C), where T = median time(days) required for the treatment-group tumors and C, the control grouptumors (median of 120 each) to reach a predetermined weight (750 mg).Tumor-free survivors were excluded from these calculations. †Control:Median day of death = 29; time for median tumor to reach 750 mg = 10.4days; there were no tumor-free survivors among the 30 control mice.‡Increase in life span, excluding survivors. δThe Log₁₀ cell kill(total) was calculated from the following formula: Log kill = T-Cvalue/(3.32 T₄). Where T₄ is the tumor volume-doubling time measuredfrom a best fit straight line of the control-group tumors in exponentialgrowth (100-400 mg range). T₄ = 1.2 for Lewis tumor in this experiment.

A third study evaluated the efficacy of IPM in the inhibition of B16melanoma growth. Single dose administrations of IPM at 150 mg revealedthat IPM was slightly inferior to CPA but better than Ifos in thisresistant animal model. There were no statistical differences between %ILS responses between the three therapeutic agents. The results of thisstudy, recorded in Table 4, demonstrate the efficacy of IPM againstmelanoma.

TABLE 4 A Comparison of the Response of s.c. B16 Melanoma toCyclophosphamide, Isophosphoramide Mustard, Phosphoramide Mustard, andIfosfamide Tumor- Rx Dosage free T-C* % Agent Schedule (mg mg⁻¹)survivors (days) ILS Cylophosphamide Day 2 200 5/10 27 68 Single doseIfosfamide Day 2 300 7/10 18 55 Single dose Phosphoramide Day 2 200 0/104.9 15 mustard Single dose Day 2 30 0/10 6.1 17 Q5 min x 7Isophosphoramide Day 2 100 6/10 8.4 34 mustard Single dose *See footnotewith Table 3, above. Predetermined weight was 750 mg.

A fourth study evaluated IPM in the inhibition of P388 Leukemia in mice.In this animal model, IPM was comparably effective to CPA and Ifosagainst IP implanted P388 leukemia as indicated by >log₁₀ cell kill,although it produced fewer tumor-free survivors. However, with theP388/CPA tumor model, there was significantly improved cell kill as wellas % ILS for IPM as compared to CPA and Ifos. The results of this studyare recorded in Table 5. All data is statistically significant anddemonstrates that IPM can be used against CPA resistant or treatedtumors as well as for patients pretreated with other agents.

TABLE 5 Activity of isophosphoramide mustard against P388/0 and P388/CPAleukaemias (Optimal response at #LD₁₀ dose, from dose-response study)P388/CPA† P388/0† Tumor burden at start of Tumor burden at start ofR_(x) = ~4.6 × 10⁶ cells R_(x) = ~4.4 × 10⁶ cells Net log₁₀ Net log₁₀Reduction reduction in % ILS in tumor Day 60 % ILS tumor burden Day 60(dying burden Dosage* survivors/ (dying after survivors/ mice afterAgent (mg kg⁻¹) total mice only) therapy‡ total only) therapy‡ CPA 2657/10 +280 7 0/10 +35 3 175 4/10 +130 7 0/10 +35 3 Ifos 538 7/10 +210 70/10 +42 4 431 7/10 +130 7 0/10 +39 4 IPM 125 0/9  +100 6 0/10 +71 7 1001/10 +140 7 0/10 +85 7 *Treatment: IP; day 1 only; highest non-toxicdose (LD₁₀ or less) in a range of doses. †Implant: IP; 10⁶ cells, infemale CDF₁ mice.

A fifth study evaluated the inhibition of implanted M5076 sarcoma withIPM in mice. IPM in doses of 18-40 mg/kg were injected IP to growingtumors daily for five days (the compound was injected IP daily on days11-15). T-C was 6.1 days at 40 mg/kg. The doses were tolerated well withsignificant improvement in response. The mice tolerated the IPtreatments well; the only toxicities are organ pathologies observed atautopsy that were associated with the induced tumors. The results ofthis study, recorded in Table 6, demonstrate that IPM is effectiveagainst sarcoma in a dose-dependent fashion.

TABLE 6 Response of SC Implanted M5076 Sarcoma to Treatment with IPMDose Days To Days Delay Agent (mg/kg) 2 doublings (T-C) IPM 40 15.4 6.1IPM 27 12.6 3.3 IPM 18 10.3 1

A sixth study evaluated the inhibition of implanted 16/C mammary tumorsin mice. Mice were implanted with the 16/C mammary tumor, and when thetumors were palpable/measurable, were treated with CPA, Ifos and IPM, asindividual agents. CPA and Ifos were used as controls for IPM. The drugswere administered IP in doses of 30-60 mg/kg/per day for 4 days,starting on day 7 after tumor implantation. There was statisticalimprovement in activity for IPM as compared to CPA and Ifos, at alldoses for the three agents. IPM was superior in ‘days to 2 doublings’and ‘days delay (T-C)’ when compared to Ifos and CPA at the samedosage/day against this aggressive murine mammary tumor. All ratios werewithin confidence limits. These data (recorded in Table 7) demonstratethe efficacy of IPM against mammary tumors and the four day dosingsfurther support the superiority of multiple dosings for IPM.

TABLE 7 Response of SC 16/C Mammary Tumor to Treatment with CPA, IFOS,and IPM Days Days Dose to 2 Delay Agent (mg/kg) Route Schedule Doublings(T-C) Control IP Q 1 dx CONTROL 3.2 4 day 7 CPA 60 IP Q 1 dx CPA 7.7 4.54 day 7 CPA 50 IP Q 1 dx 7.2 4.0 4 day 7 CPA 40 IP Q 1 dx 4.4 1.2 4 day7 CPA 30 IP Q 1 dx 3.6 0.4 4 day 7 IFOS 60 IP Q 1 dx IFOS 4.6 1.4 4 day7 IFOS 50 IP Q 1 dx 4.9 1.7 4 day 7 IFOS 40 IP Q 1 dx 3.8 0.6 4 day 7IFOS 30 IP Q 1 dx 4.0 0.8 4 day 7 IPM 50 IP Q 1 dx IPM 9.5 6.3 4 day 7IPM 40 IP Q 1 dx 8.5 5.2 4 day 7 IPM 30 IP Q 1 dx 7.4 4.2 4 day 7

A seventh study evaluated IPM against IP implanted human lox-IMVImelanoma. Nude mice were implanted IP with the human Lox melanoma andtreated for five days with either CPA or IPM. Doses for both were 40mg/kg daily IV×5 days. % ILS was +121 for CPA and +52 IPM. However,excellent responses were seen and doses were well tolerated. Responseswere within confidence levels. The results of this study (recorded inTable 8) demonstrate the efficacy of IV administration of IPM andfurther demonstrates the efficacy of IPM against human melanoma.

TABLE 8 Treatment: IV; Q1DX5 (1) Therapeutic Response Dosage Median DayAgent (mg/kg/dose) of Death % ILS Control — 19.0 — CPA 40 42.0 +121 IPM40 29.0  +52

An eighth study evaluated the inhibition of human MX-1 mammary tumorswith IPM. Daily IP administration of CPA, Ifos or IPM was compared in40-60 mg/kg dosing on a schedule of daily×5 days beginning with day 12(after implantation). The data recorded in Table 9 demonstrate that IPMis active against human mammary tumors. All ratios were in competencelimits.

TABLE 9 Response of SC MX-1 Mammary Tumor to Treatment with CPA, IFOS,and IPM Dose Days to Days Delay Agent (mg/kg) Route Schedule 2 doublings(T-C) CPA 60 IP Q 1dx 5 day 12 >48.0 >40.5 IFOS 60 IP Q 1dx 5 day 1239.4 31.9 IFOS 40 IP Q 1dx 5 day 12 16.1 8.6 IPM 60 IP Q 1dx 5 day 1226.5 19.0 IPM 40 IP Q 1dx 5 day 12 14.2 6.7

Example 5

This example compares the efficacy of IPM and that of IPM.(LYS)₂ andIPM.(NH₄)₂ salt against various hyperproliferative cell lines.

The efficacy of IPM and IPM.(LYS)₂ salt and IPM.(NH₄)₂ salt againstLewis lung murine tumor was compared when the compounds wereadministered by IP routes daily for 5-days in doses of 20-125 mg/kgdaily×5 days, beginning with day 6 (after implantation). IPM and itslysine salt possessed equivalent activities at doses that reflected a2-fold increase for the MTD (mg/kg/dose) of the salt over parent drug.All ratios were within confidence limits. The mice tolerated IPadministration of the salt well; the only toxicities are organpathologies observed at autopsy that were associated with the inducedtumors. The results of this study (recorded in Table 10) demonstratethat IPM.(LYS)₂ exhibits equivalent efficacy to IPM against Lewis lungmurine tumor, and that the IPM.(NH₄)₂ salt is effective against Lewislung murine tumor.

TABLE 10 Lewis Lung Murine Tumors MTD Dosage T-C Agent (mg/kg/dose)(days) IPM lysine salt 93.2 8.3* IPM ammonium salt 42.8 9.1 IPM 40.012.5* Implant: 20-30 mg tumor fragments Treatment Route: IntraperitonealSchedule: q1d x 5 starting day 6 *Although the T-C values arestatistically different (P = 0.004), the antitumor activities arecomparable.

A second comparison of the efficacy of IPM, IPM.(LYS)₂ salt andIPM.(NH₄)₂ salt was conducted with respect to inhibition of MX-1 mammarytumors. In this study, the effects of IPM and IPM.(LYS)₂ salt werecompared when administered IP, in doses of 20-100 mg/kg daily×5 days,beginning with day 12 following implantation of MX-1 mammary tumors inmice. IPM.(LYS)₂ salt was 8-fold superior to IPM at comparable dosing.The MTD was also higher for the lysine salt. All ratios were withinconfidence limits. The mice tolerated the IP treatment with IPM.(LYS)₂and IPM.(NH₄)₂ salts well; the only toxicities are organ pathologiesobserved at autopsy that were associated with the induced tumors. Thisdata (recorded in Table 11) demonstrates that both IPM.(LYS)₂ salt andIPM.(NH₄)₂ salt are significantly superior to IPM against human breasttumor cells.

TABLE 11 MX-1 Human Breast Tumor MTD Dosage T-C Agent (mg/kg/dose)(days) IPM lysine salt 93.2 10.2* IPM ammonium salt 28.6 4.6 IPM 40.02.1* Implant: 20-30 mg tumor fragments subcutaneously in the mammary fatpad Treatment Route: Intraperitoneally Schedule: q1d x 5 starting day 12*P-value = 0.041

Example 6

This example describes the evaluation the acute toxicity ofisophosphoramide mustard lysine salt, following three days of dailyintravenous (bolus) injection in mice. This study consisted of twophases.

First, the dose range-finding phase consisted of four treatment groups(one mouse/sex/group) that received the test article as a single dailydose for three consecutive days at respective dose levels of 100, 200,400, and 600 mg/kg. The vehicle was 0.9% sodium chloride for injection,USP and all doses were at a constant volume of 15 mL/kg. The animalswere observed for seven days following dosing. On Day 10, following theseven-day observation period, all surviving dose range-finding phase arepresented in Appendix F of this report. Based on the deaths noted in thedose range-finding phase at 200, 400, and 600 mg/kg, the dose levelschosen for the main study phase were 50, 75, 100, 200, 300, 500, and 600mg/kg (see below).

The second, main study phase consisted of eight treatment groups (fivemice/sex/group) that received the test article as a single daily dosefor three consecutive days at respective dose levels of 50, 75, 100,200, 300, 400, 500, and 600 mg/kg. An additional group (5 mice/sex)served as a parent compound control and received the isophosphoramidemustard parent compound in the same manner, at a dose level of 150mg/kg. The vehicle was 0.9% sodium chloride for injection, USP and alldoses were at a constant volume of 15 ml/kg. The animals were observedfor 11 days following the three-day dosing period.

Observations for mortality, morbidity, and the availability of food andwater were conducted twice daily for all animals. Observations forclinical signs were conducted daily during the study (approximately oneand four hours postdose on Days 1, 2, and 3, and once daily onnon-dosing days). Body weights for all surviving animals were measuredand recorded the second day after receipt, prior to randomization, andon Days −1 and 7. Body weights also were measured on all surviving mainstudy phase animals on Day 14. Macroscopic evaluations were performed oneach main study animal at necropsy (Day 15).

Animal Acquisition and Acclimation:

A total of 62 male and 61 female Crl: CD-1(1CR) BR mice (approximatelysix weeks old) were received from Charles River Laboratories, Portage,Mich., on Apr. 21, 2003. During the seven- to 16-day acclimation period,the sex of the animals was verified, the animals were weighed andobserved twice daily with respect to general health and any signs ofdisease. At receipt, the animals were housed three to four mice/cage inorder to acclimate to the automatic watering system. Three days afterreceipt, the animals were housed individually. All animals were given adetailed clinical observation prior to selection for study.

Randomization, Assignment to Study, and Maintenance:

Prior to assignment to study, the mice were weighed and examined forevidence of disease and other physical abnormalities. Animals assignedto the study had body weights within 20% of the mean body weight foreach sex. Using a simple randomization procedure, the animals wereplaced into the treatment groups. Extra animals obtained for this studywere euthanized via carbon dioxide inhalation and discarded.

Forty-nine male and 49 female mice (weighing 24.8 to 29.1 g and 21.5 to24.2 g, respectively, at randomization) were assigned to the treatmentgroups identified in Table 12.

Each animal was assigned an animal number to be used in Provantis™ andwas implanted with a microchip bearing a unique identification number.The individual animal number, implant number, and study number compriseda unique identification for each animal. The cage was identified by theanimal number, study number, group number, and sex. Animalidentification was verified during the course of the study as documentedin the data.

The animals were individually housed in suspended, stainless steel,wire-mesh type cages. Fluorescent lighting was provided forapproximately 12 hours per day and controlled via an automatic timer.Temperature and humidity were monitored and recorded daily, andmaintained between 68 to 74° F. and 30 to 68%, respectively.

The dose levels for the dose range-finding phase were selected on thebasis of available data from previous studies. The dose levels for themain study phase were set following a review of the results from thedose range-finding phase, with the exception of the 150 mg/kg parentcompound control group, whose dose level was selected on the basis ofavailable data from previous studies.

TABLE 12 Group Assignments Group Dose Level Number of Animals Number(mg/kg) Male Female Dose Range-finding Phase^(a)  1 100 1 1  2 200 1 1 3 400 1 1  4 600 1 1 Main Study Phase^(b)  5^(c) 150 5 5  6 50 5 5  775 5 5  8 100 5 5  9 200 5 5 10 300 5 5 11 400 5 5 12 500 5 5 13 600 5 5^(a)Animals were dosed for three days, followed by a seven-dayobservation period. ^(b)Animals were dosed for three days, followed by a11-day observation period. ^(c)This group was dosed withIsophosphoramide Mustard Parent Compound (Parent Compound Control).

Administration:

Four range-finding treatment groups (one mouse/sex/group) received thetest article as a single daily dose for three consecutive days viaintravenous (bolus) injection at respective dose levels of 100, 200,400, and 600 mg/kg. All doses were at a volume of 15 mL/kg and based onthe most recent body weights.

Eight main study treatment groups received the test article as a singledaily dose for three consecutive days via intravenous (bolus) injectionat respective dose levels of 50, 75, 100, 200, 300, 400, 500, and 600mg/kg. An additional group (five mice/sex) served as a parent compoundcontrol and received the Isopbhosphoramide Mustard Parent Compound inthe same manner at a dose level of 150 mg/kg. All doses were at a volumeof 15 mL/kg and based on the most recent body weight.

While the animal was restrained, the dosing formulation was administeredthrough a needle that was inserted into the tail vein and the hub of theneedle was observed for the presence of blood to ensure the properplacement of the needle in the vein. The dose was then administered atthe absolute dose volume for each animal.

Observation and Examination:

All mice were observed for morbidity, mortality, injury, and theavailability of food and water twice daily throughout the duration ofthe study.

A detailed clinical examination of each animal was performed at one andfour hours postdose on Days 1, 2, and 3, and once daily on non-dosingdays. The observations included, but were not limited to, evaluation ofthe skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, externalgenitalia, limbs and feet, respiratory and circulatory effects,autonomic effects such as salivation, and nervous system effectsincluding tremors, convulsions, reactivity to handling, and bizarrebehavior.

Body weights for all surviving animals were measured and recorded thesecond day after receipt, prior to randomization, and on Days −1 and 7.Body weights also were measured on all surviving main study phaseanimals on Day 14. The body weights recorded after receipt and prior torandomization are not reported, but are maintained in the study file.

On Day 10, all surviving dose range-finding phase animals wereeuthanized and discarded. No necropsies were conducted on any doserange-finding animals. All main study animals received a completenecropsy examination under procedures approved by a veterinarypathologist. At the termination of the study, all surviving main studyphase animals were euthanized by carbon dioxide inhalation andexsanguination via abdominal vena cava.

Each animal was examined carefully for external abnormalities includingmasses. The skin was reflected from a ventral midline incision and anysubcutaneous abnormalities were identified and correlated withantemortem findings. The abdominal, thoracic, and cranial cavities wereexamined for abnormalities and the organs were removed and examined. Allabnormalities were recorded. No tissues were saved and the carcasseswere discarded.

Statistics:

When appropriate, the LD₅₀ and the LD₁₀ and their 95% confidence limitswere calculated using the Probit Procedure (SAS Institute, Inc.SAS/STAT® User's Guide, Version 6, Fourth Edition, Volume 2. Cary N.C.:SAS Institute; 1989) in SAS® (main study treated groups).

The computer systems used during the conduct of this study are presentedin Table 13.

TABLE 13 Computer Systems In-life System: Provantis ™ Randomization:Provantis ™ Pathology: Provantis ™ Statistical Analyses: SAS Reporting:SAS and Microsoft Office Professional

Results:

The following data are the results of the definitive main study phase.

A summary of mortality results is presented in the Table 14 below. Themortality results generally exhibit a typical dose-response effect, withIPM Lysine Salt being slightly more toxic in females than in males. TheIPM parent compound control group exhibited the expected mortality, aswell as greater toxicity in females than males, correlating withavailable data from previous studies.

TABLE 14 Mortality by Day of Study and Cumulatively Study Day(Male/Female) Cumulative Dose Level 1 to 5 6 7 8 9 10 11 12 to 14Mortality (mg/kg) M F M F M F M F M F M F M F M F M F Total 150^(a) 0 00 1 0 1 0 2 0 0 0 0 1 0 0 0 1/5 4/5  5/10 50 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0/5 0/5  0/10 75 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 0/5  0/10 100 00 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1/5 0/5  1/10 200 0 0 0 0 0 0 0 0 0 0 0 20 0 0 1^(b) 0/5 3/5  3/10 300 0 0 0 1 1 3 1 0 0 0 1 1 1 0 0 0 4/5 5/5 9/10 400 0 0 0 4 3 1 1 0 0 0 0 0 1 0 0 0 5/5 5/5 10/10 500 0 0 4 2 1 30 0 0 0 0 0 0 0 0 0 5/5 5/5 10/10 600 0 0 3 5 2 0 0 0 0 0 0 0 0 0 0 05/5 5/5 10/10 ^(a)Parent Compound Control ^(b)Death occurred on Day 12M-Male F-Female

The intravenous LD₁₀ of IPM Lysine Salt was calculated to be 133 mg/kg(95% confidence limits of 65 to 172) in mice (combined sexes), while theintravenous LD₅₀ was calculated to be 220 mg/kg (95% confidence limitsof 184 to 265 mg/kg).

The LD₁₀ values for males and females separately were 140 and 179 mg/kg,respectively (95% confidence limits of 12 to 199 mg/kg for males; couldnot be calculated for females), while the LD₅₀ values for males andfemales were 247 and 197 mg/kg, respectively (95% confidence limits of187 to 330 for males; could not be calculated for females).

No treatment-related macroscopic findings were noted in either sex inpostmortem observations.

Conclusions:

Mortality results generally displayed a typical dose-response effect,with IPM Lysine Salt being slightly more toxic in females than in males.No animals died at 50 or 75 mg/kg, 1 of 10 animals died at 100 mg/kg, 3of 10 animals died at 200 mg/kg, 9 of 10 animals died at 300 mg/kg, andall animals died at 400, 500, and 600 mg/kg. The IPM parent compoundcontrol group exhibited the expected mortality (5 of 10 animals died),as well as greater toxicity in females than males, correlating withavailable data from previous studies. The onset of death on study wasslightly delayed, with the first mortalities occurring on Day 6 and thelast on Day 12. Clinical signs generally reflecting the deterioratingstate of mice prior to death were observed in both sexes. Those clinicalsigns included moribundity, decreased activity, increased activity,swelling (tail, nose/muzzle, and/or face), breathingrapid/slow/shallow/difficult/audible, tremors, skin cold to touch,unkempt appearance, posture hunched, limb function impaired, hairdiscoloration in the dorsal and/or anogenital regions, feces few/absent,and urination decreased. Treatment-related decreases in mean body weightgain, or in may cases body weight loss, were noted in surviving animalsby Day 7, with at least partial recovery by Day 14 in those animalssurviving to study termination. No treatment-related macroscopicfindings were noted at necropsy.

Based on the condition and findings of this study, the intravenous LD₁₀of IPM 2Lys was calculated to be 133 mg/kg (95% confidence limits of 65to 172) in mice (combined sexes), while the intravenous LD₅₀ wascalculated to be 220 mg/kg (95% confidence limits of 184 to 265 mg/kg).

Example 7

This example summarizes the results of extensive pro-clinical data forthe toxicity of IPM and its lysine salt. This data is used to designdosage regimens for human clinical trials.

The toxicity of IPM and its lysine salt have been investigated throughpre-clinical acute and sub-acute studies using mice, rats and dogs.Single dose oral, intravenous (IV) and intraperitoneal (IP) routes ofadministration for IPM have been studied in mice and rates. Multipledaily dose administrations—IV and IP—have been studied in mice and dogs.Sub-acute intravenous (3-day) dosing in the mouse and dog has providedthe toxicology/pharmacokinetic information regarding toxicities and drugdisturbances that were utilized in designing the administration and doseschedules in humans. Sub-acute IV (3-day) dosing with the IPM lysinesalt was conducted in the mouse.

Based upon the results of the dose range finding study, higher doses ofIPM were required to produce mortality than anticipated. For rats, theoral LD₅₀ values were calculated to be 4443 mg/kg for males, 2786 mg/kgfor females and 3560 mg/kg for both sexes combined. In each case, the95% confidence limits could be calculated.

For mice, oral LD₅₀ values were calculated to be 1014 mg/kg for males(95% confidence limits), 1962 mg/kg for females (95% confidence limitsof 1523-2983 mg/kg) and 1432 mg/kg for both sexes combined (95% limitsof 1128-1742 mg/kg).

For rats, single dose intravenous LD₅₀ values were calculated to be 567mg/kg for males, 400 mg/kg for females and 428 mg/kg for both sexescombined. In each case, the 95% confidence limits could not becalculated. For mice, intravenous LD₅₀ values were calculated to be 929mg/kg for males (95% confidence limits), 484 mg/kg for females (95%confidence limits of 72-1364 mg/kg) and 688 mg/kg for both sexescombined (95% confidence limits of 398-1366 mg/kg).

Administration of IPM by IV and IP routes did result in acute deaths formice, rats and dogs. Oral administration to mice and rats was alsoevaluated and LD₅₀ values were determined in the 1.4-3.5 g/kg range forthese rodent species. Acute intravenous toxicity symptoms in mice, ratsand dogs, included less appetite, diarrhea, decreased activity anddeath.

The acceptable doses from the three (3) day dosing studies weresignificantly different from the single dose schedule. The effects ofthe drug on bone marrow, spleen and renal tubular functions wereevaluated. The impact of IPM on these organs appears to contribute tothe cause of death in these two species. A summary is presented below.

A sub-acute IV study of IPM in mice provided information as to LD₁₀values and toxicity that could occur in humans. The mortality resultsdisplayed a typical dose-response effect, with IPM being slightly moretoxic in females than in males.

The intravenous LD₁₀ of IPM was calculated to be 119 mg/kg (with 95%confidence limits of 87-134 mg/kg) in mice (combined sexes), while theintravenous LD₅₀ was calculated to be 149 mg/kg (with 95% confidencelimits of 132-169 mg/kg). The LD₁₀ values for males and femalesseparately were 168 and 125 mg/kg, respectively, while the LD₅₀ valuesfor males and females were 176 and 132, respectively. In each case, the95% confidence limits could not be calculated.

The sub-acute IPM lysine salt study included a total of 40 male and 40female mice (Crl: CD-1(1CR)BR) weighing 24.8 to 29.1 g and 21.5 and 24.2g, respectively, at randomization) were treated with doses of 50 to 600mg/kg IV daily×3 days (Table 8.8).

For IPM LYS salt, the intravenous LD₁₀ for the 3-day mouse study wascalculated to be—133 mg/kg (95% confidence limits 65 to 172 mg/kg(combined sexes)), while the intravenous LD₅₀ was 220 mg/kg (with 95%confidence limits of 184 to 265 mg/kg (for combined sexes)). The LD₁₀values for males and females separately were 140 and 179 mg/kg,respectively (95% confidence limits of 12 to 199 mg/kg for males; couldnot be calculated for females). The LD₅₀ values for males and femaleswere 247 and 197 mg/kg, respectively (95% confidence limits of 187 to330 for males; could not be calculated for females).

The IPM lysine salt generally displayed a typical dose-response effect,with slightly more toxicity seen in females. No mice died at 50, 75 or200 mg/kg, 1 of 10 animals died at 100 mg/kg, 9 out of 10 animals diedat 300 mg/kg, and all mice died at 400, 500, and 600, mg/kg. The parentIPM control group exhibited the expected mortality, as well as greatertoxicity in females than males, correlating with available data fromprevious studies. The onset of death on study was slightly delayed, withthe first mortalities occurring on Day 6 and the last on Day 12.Clinical signs generally reflecting the deteriorating state of miceprior to death were observed in both sexes.

Based on the findings of microscopic examination, IPM administered aloneor as the lysine salt IV daily for three days produced treatment-relatedbone marrow depletion, kidney tubular necrosis, or a combination of bothand were considered the cause of death. For IPM, severe bone marrowdepletion was present in males at 178 mg/kg and higher, and in femalesat 133 mg/kg and higher. Kidney tubular necrosis occurred in males at237 mg/kg and higher, and in females at 133 mg/kg and higher. Inaddition, splenic lymphoid depletion was noted in most males and in allfemales that died during the study. No obvious treatment-relatedmicroscopic findings were noted in either sex at 75 mg/kg. Clinicalsigns generally secondary to the deteriorating state of the mice priorto death were observed but no clear evidence of body weight effects wereseen in mice surviving to study termination.

The intravenous LD₁₀ for isophosphoramide mustard (IPM) and its lysinesalt administered daily for three days were calculated to be 119 mg/kgvs. 133 mg/kg, respectively, with LD₅₀ calculated as 149 mg/kg vs. 220mg/kg, respectively.

Acute and sub-acute toxicity studies in rodents and dogs have beenperformed with IPM and its lysine salt. These studies also have beenused to develop acceptable starting doses human investigations. Asummary of the rodent and dog toxicity data for IV administration of IPMis recorded in Table 15 and a summary of the mouse toxicity data for IVadministration of IPM.(LYS)₂ is recorded in Table 16.

TABLE 15 Summary of Intravenous Treatment Experience - IPM Total Safety# and Dose, Regimen Dose of Plasma LD₅₀ Specie and Duration IPM (mg) IPMEfficacy (mg/kg) 47 400-2,000 mg/kg; 81.6-650 Not Not 428 Rats Iv x 1 dTested Tested 40 100-1,200 mg/kg; 3.2-36 Not Not 688 Mice Iv x 1 dTested Tested 80 75-562 mg/kg;  5.7-60.6 Not Not 149 Mice iv daily x 3days Tested Tested 14 1-100 mg/kg/d;  22.5-2130 100 mg/kg/day x Not 1-5mg/kg/day x Dogs iv daily x 3 days 3 days Tested 3 days (C_(mzx)25-78mcg/ml) (100% survival)

TABLE 16 Intravenous IPM Lysine Salt Total Safety # and Dose, RegimenDose of Plasma LD₅₀ Specie and Duration IPM (mg) IPM Efficacy (mg/kg) 8050-600 mg/kg/d; 4.3-80 Not Not 220 Mice iv daily x Tested Tested 3 days

The IPM MTD for dogs was 5 mg/kg/day×3 days and a correspondencestarting dose in humans of 100 mg/m² per day for three (3) days shouldbe a safe starting point. For IPM.(LYS)₂, the LD₁₀ for the intravenousthree (3) day dose schedule in the mouse was calculated to be—133mg/kg/day×3 days. IPM.(LYS)₂ is considered to be a minimally toxicalkylating agent with a steep therapeutic range. On mg/kg basis, themean toxic dose (MTD) in humans for the lysine salt is estimated as 1/10the LD₁₀ in mice or 40 mg/m²/d.

Estimated comparable human IV dosages are recorded in Table 17.

TABLE 17 Estimated Comparable Human Intravenous Dosages Comparable DrugSpecies Sub-acute IV LD₁₀ Human IV Dosage IPM Mouse 119 mg/kg/d 30mg/m²/d IPM Dog  5 mg/kg/d 100 mg/m²/d  IPM Lysine Salt Mouse 133mg/kg/d 40 mg/m²/d

Example 8

This example describes the treatment of cancer in human subjects havingmetastatic ovarian cancer.

The subject is treated with IPM 500 mg/m² daily for three consecutivedays via intravenous infusion. Her serum electrolytes, such asphosphorus and chloride are corrected with supplemental electrolytes,which are discontinued after seven days. BUN and creatinine aremonitored normal limits.

Example 9

This example describes the results of treating human subjects withIPM.(LYS)₂. To date four (4) patients with advanced cancer have beentreated with IPM.(LYS)₂.

The initial dose of IPM lysine salt was 30 mg/m² was administratedintravenously daily for three (3) days. One patient (cohort) was treatedper dose escalation every 21-28 days to allow for toxicity presentation.Doses were escalated by 40% if there were no serious toxic events. Fourpatients have been treated—one at each dosage—30, 42, 59 and 83 mg/m²via daily IV administration for 3 days without serious toxicity. Onepatient with rectal cancer had stabilization of his disease followingadministration of 83 mg/m² of IPM.(LYS)₂ via daily IV administration for3 days.

Example 10

This example describes the treatment of cancer non-small cell lungcancer which has progressed to metastatic infiltrating moderatelydifferentiated adenocarcinoma. The status of the disease can beconfirmed by CAT scan.

Isophosphoramide mustard lysine salt is administered at 350 mg/m² dailyfor three consecutive days intravenously. After a 21-day rest period,the three-day treatment protocol is repeated once. Daily blood fluidchemistry and hematological studies are monitored during treatments. Thestatus of the cancer is monitored by CAT scan.

Example 11

This example demonstrates the effect of formation of an amine salt oncompound stability.

Samples of lyophilized isophosphoramide mustard and its lysine salt werestored under varying conditions and assayed for purity. Results arepresented in the table below:

Compound 0 months 1 months 3 months 1 year IPM, −23° C. 97% 88% 70%IPM-LYS, −23° C. 98% 98% 100% IPM-LYS, ambient 98% 98% 65%

1. A compound of the formula:

wherein A⁺ represents an ammonium species selected from the groupconsisting of quaternary ammonium, the conjugate acid of a basic aminoacid, acyclic aliphatic ammonium, dialkyl ammonium, trialkyl ammonium,heterocyclic ammonium, aromatic ammonium, substituted and unsubstitutedpyridinium, guanidinium, and amidinium; and X and Y independentlyrepresent leaving groups.
 2. A compound of claim 1, wherein A⁺represents BH⁺ and B is an amine selected from the group consisting ofthe basic amino acids, pyridine, N,N-dimethylaminopyridine,diazabicyclononane, diazabicycloundecene, N-methyl-N-ethylamine,diethylamine, triethylamine, diisopropylethylamine, mono-, bis- ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine.
 3. A compound ofclaim 2, wherein the compound has the formula

and G represents a second ammonium or amine species.
 4. A compound ofclaim 2, wherein B is selected from the group consisting of lysine,homolysine, arginine, homoarginine, histidine, ornithine andcombinations thereof.
 5. A compound of claim 2, wherein B is lysine. 6.A compound of claim 2, wherein X and Y independently are selected fromthe group consisting of the halogens and the sulfonates.
 7. A compoundof claim 2, wherein X and Y are halogen.
 8. A compound of claim 2,wherein X and Y are chlorine.
 9. A compound of claim 3, wherein B and Gare lysine and X and Y represent chlorine.
 10. A salt comprising anisophosphoramide mustard anion and an ammonium cation, wherein theammonium cation is selected from the group consisting of quaternaryammonium, the conjugate acid of a basic amino acid, acyclic aliphaticammonium, dialkyl ammonium, trialkyl ammonium, heterocyclic ammonium,aromatic ammonium, substituted and unsubstituted pyridinium,guanidinium, and amidinium.
 11. A salt of claim 10 further comprising asecond amine or ammonium cation.
 12. A salt of claim 10, wherein theammonium cation is the conjugate acid of lysine ortris(hydroxymethyl)methylamine.
 13. A pharmaceutical compositioncomprising a compound of claim 1 and a pharmaceutically acceptablecarrier.
 14. A pharmaceutical composition of claim 13, wherein X and Yindependently are selected from the group consisting of the halogens andthe sulfonates.
 15. A pharmaceutical composition comprising a compoundof claim 2 and a pharmaceutically acceptable carrier.
 16. Apharmaceutical composition of claim 13, wherein X and Y are halogen. 17.A pharmaceutical composition of claim 13, wherein X and Y are chlorine.18. A pharmaceutical composition of claim 13, wherein the compositioncomprises a solution formulated for administration to a human subject.19. A pharmaceutical composition of claim 18, wherein the solutioncomprises from about 0.1 mg/mL to about 250 mg/mL of the compound.
 20. Apharmaceutical composition of claim 19, wherein the solution comprisesfrom about 20 to about 100 mg/mL of the compound.
 21. A pharmaceuticalcomposition of claim 13, wherein the composition comprises from about200 mg to about 1500 mg of the compound per dosage unit.
 22. Apharmaceutical composition comprising a compound of claim 3 and apharmaceutically acceptable carrier.
 23. A pharmaceutical compositioncomprising a compound of claim 9 and a pharmaceutically acceptablecarrier.
 24. A pharmaceutical composition comprising a salt ofisophosphoramide mustard and an amine base; and a pharmaceuticallyacceptable carrier, wherein the amine base is selected from the groupconsisting of ammonia, a basic amino acid, acyclic aliphatic amine,dialkyl ammonium, trialkyl ammonium, heterocyclic amine, aromatic amine,substituted and unsubstituted pyridine, guanidine, and amidine.
 25. Apharmaceutical composition of claim 24, wherein the compositioncomprises at least two equivalents of the amine base per equivalent ofisophosphoramide mustard.
 26. A pharmaceutical composition of claim 24,wherein the amine base is selected from the group consisting of basicamino acids, acyclic aliphatic amines, di- and tri-cyclicalkyl amines,heterocyclic amines, aromatic amines, substituted and unsubstitutedpyridines, guanidines, and amidines.
 27. A pharmaceutical composition ofclaim 24, wherein the amine base is selected from the group consistingof lysine, homolysine, arginine, homoarginine, histidine, ornithine andcombinations thereof.
 28. A composition according to claim 24, whereinthe amine base is lysine or tris(hydroxymethyl)methylamine.
 29. Alyophilisate comprising a compound of claim 1, produced by a methodcomprising contacting a compound of the formula

with at least one equivalent of an amine base selected from the groupconsisting of the basic amino acids, acyclic aliphatic amines, dialkylammonium, trialkyl ammonium, heterocyclic amines, aromatic amines,substituted and unsubstituted pyridines, guanidines, and amidines; inthe presence of water, and lyophilizing the resulting mixture, therebyforming the lyophilisate.
 30. A lyophilisate of claim 29, wherein X andY are chlorine.
 31. A lyophilisate of claim 29, wherein the lyophilisateis formed with at least two equivalents of the amine base.
 32. Alyophilisate of claim 29, wherein the amine base is lysine ortris(hydroxymethyl)methylamine.
 33. A compound of claim 2, wherein thecompound is an anti-hyperproliferative compound.
 34. A compound of claim2, wherein the compound is more effective against CPA-resistant tumorgrowth than CPA, Ifos, or both.
 35. A compound of claim 2, wherein B istris(hydroxymethyl)methylamine.
 36. A compound of claim 35, wherein Xand Y are halogen.
 37. A compound of claim 36, wherein X and Y arechlorine.
 38. A pharmaceutical composition comprising the compound ofclaim 35 and a pharmaceutically acceptable carrier.
 39. A pharmaceuticalcomposition of claim 38, wherein the composition comprises a solutionformulated for administration to a human subject.
 40. A pharmaceuticalcomposition of claim 15, wherein B is selected from the group consistingof lysine, homo lysine, arginine, homoarginine, histidine, ornithine andcombinations thereof.
 41. A pharmaceutical composition of claim 38,wherein the compound is a lyophilisate.
 42. A compound of claim 13,wherein the compound is a lyophilisate.
 43. A sterile aqueous solutionsuitable for administration to a human patient, comprising a compounddissolved directly therein of the formula:

wherein A⁺ represents an ammonium species selected from the groupconsisting of quaternary ammonium, the conjugate acid of a basic aminoacid, acyclic aliphatic ammonium, dialkyl ammonium, trialkyl ammonium,heterocyclic ammonium, aromatic ammonium, substituted and unsubstitutedpyridinium, guanidinium, and amidinium; and X and Y independentlyrepresent leaving groups.
 44. A sterile aqueous solution suitable foradministration to a human patient, having dissolved directly therein(i). a compound of the formula:

wherein X and Y independently represent leaving groups, and (ii). anammonium species selected from the group consisting of quaternaryammonium, the conjugate acid of a basic amino acid, acyclic aliphaticammonium, dialkyl ammonium, trialkyl ammonium, heterocyclic ammonium,aromatic ammonium, substituted and unsubstituted pyridinium,guanidinium, and amidinium; wherein the compound in the solution has ahalf-life at room temperature that is equal or greater to five times thehalf-life of the compound in solution in the absence of the ammoniumspecies.
 45. A sterile lyophilisate of a compound of the formula:

wherein A⁺ represents an ammonium species selected from the groupconsisting of quaternary ammonium, the conjugate acid of a basic aminoacid, acyclic aliphatic ammonium, dialkyl ammonium, trialkyl ammonium,heterocyclic ammonium, aromatic ammonium, substituted and unsubstitutedpyridinium, guanidinium, and amidinium; and X and Y independentlyrepresent leaving groups, and wherein the lyophilisate is formed from asterile solution of the compound dissolved in water.
 46. Thelyophilisate of claim 45, wherein A⁺ represents an acyclic aliphaticammonium that includes one or more substitutions with a hydroxyl group;and X and Y independently are selected from the halogens and thesulfonates.
 47. A lyophilisate of claim 46, wherein the acyclicaliphatic ammonium is selected from the group consisting of basic aminoacids, mono-(2-hydroxyethyl)amine, bis-(2-hydroxyethyl)amine,tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,tris(hydroxymethyl)methylamine, N,N-dimethyl-N-(2-hydroxyethyl)amine,tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine.
 48. A lyophilisateof claim 46, wherein the acyclic aliphatic ammonium istris(hydroxymethyl)methylammonium, and X and Y are each chlorine.
 49. Alyophilisate of claim 29, wherein the compound in the lyophilisate doesnot substantially undergo decomposition during storage of thelyophilisate for ten (10) months at −20° C.